The present invention relates to 16-hydroxy-9Z,12Z,14E-octadecatrienoic acid and closely related compounds, such as simple esters, amides, triglycerides, or other derivatives of the carboxylic acid function; isolated from the carbonate fraction of free acids extractable from various plant materials, particularly alfalfa. The invention also relates to compositions, containing the compounds, which are used for retarding red blood cell sickling associated with sickle cell disease.
Sickle cell disease is an inherited disease stemming from inadequate oxygen transport by an abnormal type of hemoglobin molecule in the red blood cells. It is an inherited disease which can be passed to offspring only if both parents carry the genetic trait. The trait carriers show no sign of the disease, but statistically one in four of their children will be afflicted with the disease. The disease is most prevalent in the black races, but is also known in other races surrounding the Mediterranean Sea and in India. It affects about 0.2% of the U.S. black population but is much more prevalent in central Africa.
The most common manifestation of the disease is an extremely painful "crisis," typically lasting several days, and affecting one or another local part of the body. The crisis often occurs following physical stress, and appears to be due to limited oxygen supply to the affected part. This is due to the inferior oxygen-carrying capability of the mutant hemoglobin, as well as to its tendency to aggregate in insoluble gels within the red blood cell, often leading to a form resembling a sickle. The distorted cells no longer freely traverse capillaries, further limiting oxygen supply to the tissues.
Despite the fact that the cause of sickle disease, i.e., the very minor structural variation in the mutant hemoglobin, has been known for many years, little progress has been made in suitable treatment of the disease. At the present time, the major treatment for the painful crises is medication for relief of pain, which merely treats the immediate symptom. Tissue damage, often involving major organs, occurs with each successive episode of oxygen deprivation, and the cumulative effects of the disease are debilitating and life shortening. Those afflicted with severe forms of the disease usually do not live through teen years.
Based on current knowledge of the disease, it appears feasible to develop a drug which will alleviate all of the symptoms of the disease and provide perfectly normal lives and life-expectancies for patients. This drug would not cure the disease, because it is genetic in origin, but if available, should effectively treat the disease by alleviation or prevention of its symptoms. The requisite capabilities of a potentially useful drug have been defined by the Sickle Cell Disease Branch of the U.S. National Institutes of Health (NIH). The requirements are specified in terms of several laboratory bioassays in which a candidate drug must perform successfully. No drug meeting these requirements has yet been announced. The drug of the present invention, however, displayed excellent responses in all the bioassays, as can be seen in the Examples below.
Fadulu (Fadulu, "Ethyl-Alcohol Extract From Fagara Zanthoxyloides Root: In vitro Effect on Red Blood Cells," Faculty Research Journal, Texas Southern University, 1:20-31 (1977)) reported in the 1970's that the extract of the African chewing stick, prepared from the roots of the tree Fagara zanthoxyloides, possessed anti-sickling properties in in vitro studies.
Chemical studies were initiated to isolate and identify the active principle(s) in the extract responsible for this activity. Systematic fractionation of the extract, coupled with bioassay of the fractions produced at each stage, led to the isolation of a small amount of a mixture of rather polar compounds.
This mixture showed good activity in the blood-agar plate test developed by Fadulu to test for anti-sickling drugs. Sheep blood is dispersed in agar in a standard agar plate. On heating in a laboratory oven at 70.degree. C. for 15 minutes, the red plates turn rust brown. If a drop of a solution of an effective anti-sickling drug is placed on the plate prior to heating, the blood under that spot remains red while the rest of the plate turns brown. The ready availability of this simple test sped the progress of the chemical work since fractions could be evaluated immediately by the chemical workers.
The chromatographic behavior of the components in the active fraction strongly suggested that they were acidic. Specifically, their TLC spots tailed badly, but were improved by the addition of a trace of acetic acid to the eluting solution. That they were indeed acids was confirmed by dissolving them in base, and then precipitating them with acid. Their presence as the free acids in the Fagara extract was confirmed in a similar experiment which resulted in their direct extraction, along with other acids, from the extract.
Because Fagara root was extremely difficult to obtain in adequate quantities, a quick survey of other plant materials was undertaken with the objective of locating a more readily available source. It was somewhat surprising to find that each of the new plant materials evaluated, primarily green vegetables, afforded the same TLC group of polar acids when extracted with dilute base. Additionally, each of the crude extracts showed good anti-sickling activity on red blood cells in vitro using the blood-agar plate test described above. Included were spinach, species spinacia oleracea, mustard greens, species Brassica juncea, grass clippings, species Stenotaphrum secundatum, alfalfa, species Medicago sativa, and even fallen oak leaves, species Quercus nigra. The greatest quantities of acids were found in alfalfa, next greatest in hay, followed by Fagara, the reference point. Lesser quantities were present in grass clippings, still less in spinach and mustard greens, and least in oak leaves. Further, alfalfa, then hay, contained greater proportions of the most polar components. The less polar acids were common stearic, linolenic, linloleic, and oleic acids, which were of no interest. The alfalfa extract contained the novel compounds both in greater quantity and proportion; additionally, because alfalfa is commercially available year-round, alfalfa was selected for further study.